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Increased Cardiovascular Risk with Pain-relieving Coxib

Increased Cardiovascular Risk with Pain-relieving Coxib Medications: Disruption of Cholesterol Metabolism. Allison B. Reiss, M.D. Vascular Biology Institute Winthrop University Hospital. The Scope of the Current Problem for Coxibs and NSAIDs and CV/GI Risk.

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Increased Cardiovascular Risk with Pain-relieving Coxib

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  1. Increased Cardiovascular Risk with Pain-relieving Coxib Medications: Disruption of Cholesterol Metabolism Allison B. Reiss, M.D. Vascular Biology Institute Winthrop University Hospital

  2. The Scope of the Current Problem for Coxibs and NSAIDs and CV/GI Risk • Given the amount of recent information in the lay press and medical journals, patients are confused about both the CV and GI effects of NSAIDs vs. coxibs and reluctant to follow physician recommendations • Physicians are reluctant to prescribe newer medication for their patients, as it takes a lot of time to evaluate the scientific evidence, data are sometimes contradictory, and they are concerned about potential legal issues, real or imagined NSAIDs=nonsteroidal anti-inflammatory drugs; GI=gastrointestinal; CV=cardiovascular.

  3. Pathological Stages of Atherogenesis • Endothelial injury • Monocyte recruitment by injured endothelium • Fatty streak formation • Fibrous plaque maturation

  4. Cholesterol is Metabolized in the Artery Wall • Not just a “pipe,” but a metabolically active organ system that maintains vascular homeostasis • An active participant in the atherosclerotic process • Endothelial dysfunction may contribute to development and clinical expression of atherosclerosis • 1989 cholesterol 27-hydroxylase, a cholesterol metabolizing enzyme, is sequenced and cloned • 27-Hydroxylase previously thought to be a liver enzyme, but…

  5. 1994: Cholesterol is Not Just Metabolized in the Liver • Reiss AB, Martin KO, Javitt NB, Martin DW, Grossi EA, Galloway AC. Sterol 27-hydroxylase: high levels of activity in vascular endothelium.J Lipid Res. 1994;35:1026-1030. • Bjorkhem I, Andersson O, Diczfalusy U, Sevastik B, Xiu R-J, Duan C, Lund E. Atherosclerosis and sterol 27-hydroxylase: evidence for a role of this enzyme in elimination of cholesterol from human macrophages.Proc Natl Acad Sci U S A. 1994;91:8592-8596.

  6. Endothelial Defense MechanismsAgainst Plaque Formation Cholesterol 27-Hydroxylase Cholesterol 27-Hydroxycholesterol

  7. Cholesterol Metabolism by the P450 Cholesterol 27-Hydroxylase Cholesterol 27-hydroxylase 27-Hydroxycholesterol Cholesterol

  8. ReverseCholesterolTransport Circulating Cholesterol (bound by plasma lipoproteins) Cholesterol ABCA1 ABCA1 Cholesterol 27-Hydroxylase LXR Cholesterol (bound by plasma lipoproteins) 27-Hydroxycholesterol Macrophage cholesterol 27-Hydroxycholesterol 27-Hydroxycholesterol Excreted as Bile Acids from the Liver

  9. Anti-Atherogenic Effects of 27-Hydroxycholesterol • Inhibition of cholesterol synthesis • Inhibition of foam cell formation • Inhibition of smooth muscle proliferation • Solubilization of cholesterol for transport to the liver • Induces expression of the cholesterol efflux molecule ABCA1 Cholesterol 27-Hydroxylase 27-Hydroxycholesterol Cholesterol

  10. Cerebrotendinous Xanthomatosis Genetically determined absence of cholesterol 27-hydroxylase leads, invariably, to the development of premature coronary artery disease Cholesterol 27-Hydroxylase Cholesterol 27-Hydroxycholesterol

  11. What Controls Vascular Expression of Cholesterol 27-Hydroxylase? • Not the obvious: bile acids, cholesterol or steroids • A hint from the immune system: autoimmune diseases such as lupus and rheumatoid arthritis are associated with an increased risk of atherosclerosis

  12. Studying Human Gene Expression in the Laboratory: THP-1 Monocytes/Macrophages • A human monocytic cell line with distinct monocytic markers isolated circa 1980 • Matures into macrophage-like adherent cells following stimulation with phorbol 12-myristate 13-acetate or phorbol dibutyrate • Derived from the peripheral blood of a 1 year old male with acute monocytic leukemia. • Widely used as in vitro model for human monocytes/macrophages

  13. IFN-g Downregulates Cholesterol27-Hydroxylase in THP-1 Cells 27-OH mRNA GAPDH Cholesterol 27-OH mRNA (% control) Control IL-1 TNF- IFN-g 27-OH Protein

  14. IC-C1q Diminish 27-Hydroxylase Message in Peripheral Blood Mononuclear Cells 27-Hydroxylase GAPDH % Control mRNA Control IC-C1q

  15. PMA Acetylated LDL In Vitro Foam Cell Formation Murine Peritoneal Macrophages Or THP-1 Monocytoid Cells Foam Cells Oil Red O

  16. IFN- Increases Foam Cell Formation in Lipid-loaded THP- 1cells Control IFN-

  17. ATP-Binding Cassette Transporter 1(ABCA1) • ABCA1 is an integral membrane protein that utilizes ATP as a source of energy for transporting lipids and other metabolites across membranes, where they are removed from cells by apolipoproteins such as apoA-I • ABCA1 is a key regulator of cellular cholesterol and phospholipid transport. • Patients with mutations in the ABCA1 gene have Tangier Disease with low levels of HDL, high levels of triglycerides and cardiovascular disease.

  18. Immune Complexes Downregulate ABCA-1 Gene Expression in THP-1 Cells Control IC

  19. Cyclooxygenase-2 (COX-2) Inhibitors: an Overview • Selective inhibitors of COX-2 are highly effective anti-inflammatory and analgesic drugs that exert their action by preventing the formation of prostanoids. • Used to relieve the symptoms of osteoarthritis and rheumatoid arthritis, and to treat dysmenorrhea.

  20. The APPROVe Trial • APPROVe trial (Adenomatous Polyp Prevention on Vioxx) • Trial on benign sporadic colonic adenomas showed a significant increase in the incidence of cardiovascular events in rofecoxib-treated subjects compared with placebo (relative risk 1.92, 95% confidence interval 1.19 to 3.11) • Vioxx (rofecoxib) was withdrawn from the market by its manufacturer, Merck, on September 30, 2004. • Decreased utilization of this highly effective class of pain relieving medications in clinical practice.

  21. COX-2 Inhibitors Elevate Heart Attack and Stroke Incidence: Why? • COX-2-derived prostacyclins exert a cardioprotective effect. • When drugs inhibit COX-2 activity, prostacyclin levels fall, leaving arteries more vulnerable to clotting. • COX-2 inhibitors suppress biosynthesis of PGI2, a potent vasodilator and platelet inhibitor without a concomitant change in the production of the prothrombotic product, thromboxane (Tx)A2. • COX-2 inhibitors raise BP more frequently than NSAIDs or placebo.

  22. Prostanoid Biosynthesis

  23. A New Theory • There is intense interest in understanding the mechanism(s) that lead to increased CV consequences of COX-2 inhibition. • COX-2 is widely expressed in atherosclerotic plaques and in the arterial wall. • Concentrations of the COX-2 protein are increased in endothelial cells, smooth muscle cells, and macrophages in human atherosclerotic lesions. • Does COX-2 inhibition affect cholesterol accumulation? • HYPOTHESIS: Pro-atherogenic mechanisms of selective COX-2 inhibition include compromise of cholesterol balance in the artery wall.

  24. Dose-Dependent Decrease in 27-Hydroxylase mRNA with COX-2 Inhibition in THP-1

  25. COX-2 Inhibition Decreases 27-Hydroxylase Protein in THP-1 Cells 1 2 3 4 Beta-Actin 27-OHase Control NS-398 NS-398 NS-398 10mM 50mM 100mM Cultured THP-1 cells were untreated or exposed to NS-398 for 18 hours. Total cell protein was isolated and 27-OHase detected with specific antibody with Beta-actin as an internal standard.

  26. COX-2 Inhibition Decreases ABCA1 mRNA in THP-1

  27. Dose-Dependent Decrease in ABCA1 Protein with COX-2 Inhibition in THP-1 Macrophages Control NS398 10 mM 50 mM 100 M ABCA1 ß-actin

  28. NS-398 Increases Foam Cell Transformation of Lipid-loaded THP-1Macrophages a b NS-398 Control Photomicrographs at 40X magnification of lipid laden THP-1macrophages stained with oil-red-O. Figure 1 . Photomicrographs at 40X magnification of lipid laden macrophages stained with oil-red-O. a) Minimal foam cell formation of THP-1 macrophages treated with acetylated LDL alone (control), compared with, b) THP-1 macrophages treated with NS-398 showing significant increase in foam cell transformation as seen by greater propensity to stain with oil-red O. Figure 1 . Photomicrographs at 40X magnification of lipid laden macrophages stained with oil-red-O. a) Minimal foam cell formation of THP-1 macrophages treated with acetylated LDL alone (control), compared with, b) THP-1 macrophages treated with NS-398 showing significant increase in foam cell transformation as seen by greater propensity to stain with oil-red O. Figure 1 . Photomicrographs at 40X magnification of lipid laden macrophages stained with oil-red-O. a) Minimal foam cell formation of THP-1 macrophages treated with acetylated LDL alone (control), compared with, b) THP-1 macrophages treated with NS-398 showing significant increase in foam cell transformation as seen by greater propensity to stain with oil-red O. Figure 1 . Photomicrographs at 40X magnification of lipid laden macrophages stained with oil-red-O. a) Minimal foam cell formation of THP-1 macrophages treated with acetylated LDL alone (control), compared with, b) THP-1 macrophages treated with NS-398 showing significant increase in foam cell transformation as seen by greater propensity to stain with oil-red O. Figure 1 . Photomicrographs at 40X magnification of lipid laden macrophages stained with oil-red-O. a) Minimal foam cell formation of THP-1 macrophages treated with acetylated LDL alone (control), compared with, b) THP-1 macrophages treated with NS-398 showing significant increase in foam cell transformation as seen by greater propensity to stain with oil-red O.

  29. Prostanoid Biosynthesis

  30. 27-OHase and ABCA1 mRNA are Decreased by NS398 and the Decrease is Reversed by PGE2 or PGD2, but Not TXA2 A) Control PGE2 PGD2 TXA2 + NS-398 B) Control PGE2 PGD2 TXA2 + NS-398

  31. Atheroma-promoting Effect of NS398 is Magnified by SLE Plasma • COX-2 inhibition in the presence of 50% SLE plasma increases THP-1 macrophage foam cell transformation significantly compared to SLE plasma alone (82.7 ± 2.8 % vs. 42.8 ± 5.3%, p<0.001). • Addition of PGD2 or PGE2 significantly reversed NS398-induced foam cell transformation in the presence of SLE plasma. • Exaggerated risk to SLE patients treated with COX-2 inhibitors, perhaps due to circulating inflammatory mediators.

  32. COX-2 RNA Silencing Decreases ABCA1 • Transfection with COX-2 siRNA significantly diminished ABCA1 message, 22.9 ± 4.9% decrease vs. mock, p=0.03. a.

  33. COX-2 RNA Silencing Promotes Foam Cell Formation • THP-1 macrophages transfected with COX-2 gene silencer had greater propensity to form lipid laden foam cells than mock transfected cells, 58.3% ± 1.6 increase, p=0.003.

  34. Acetaminophen Exposure Does Not Affect RCT Genes

  35. SUMMARY: COX Inhibition and Reverse Cholesterol Transport • COX inhibition decreases expression of cholesterol 27-OHase and ABCA1. • COX inhibition also increases foam cell transformation in THP-1 macrophages. • Observed increases in cardiovascular risk with COX inhibition may be ascribed at least in part to altered cholesterol metabolism. • Notably, disrupted cholesterol balance may be corrected by specific PGs, suggesting possibile future therapeutic modalities for COX inhibition that would support an anti-atherogenic PG state. • Signal transduction pathways involved in prostaglandin-mediated effects on 27-OHase levels remain to be elucidated.

  36. Acknowledgements Kamran Anwar, Ph.D. Sari D. Edelman, D.O. Edwin S. L. Chan, M.D. Steven Carsons M.D. Arthritis Foundation, S.L.E. Foundation, AHA, NIH

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